Design and Synthesis of Phospholipase C and A2-Activatable Near-Infrared Fluorescent Smart Probes

摘要

The primary focus of this work was to develop activatable probes suitable for in vivo detection of phospholipase activity. Phospholipases (PLs) are ubiquitous enzymes that perform a number of critical regulatory functions. They catalyze phospholipid breakdown and are categorized as A,, A2 (PLA2), C (PLC), and D (PLD) based on their site of action. Here, we report the design, synthesis, and characterization of self-quenching reporter probes that release fluorescent moieties upon cleavage with PLA2 or PLC. A series of phospholipids were synthesized bearing the NIR fluorophore pyropheophorbide a (Pyro) at the sn-2 position. Fluorescence quenching was achieved by attachment of either a positively charged black hole quencher-3 (BHQ-3) to the phospholipid headgroup or another neutral Pyro moiety at the sn-1 position. The specificity to different phospholipases was modulated by insertion of spacers (C6, C_(12)) between Pyro and the lipid backbone. The specificity of the quenched fluorescent phospholipids was assayed on a plate reader against a number of phospholipases and compared with two commercial probes bearing the visible fluorophore BODIPY. While PyroC6-PyroC6-PtdCho revealed significant background fluorescence, and a 10% fluorescence increase under the action of PLA2, Pyro-PtdEtn-BHQ demonstrated high selective sensitivity to PLC, particularly to the PC-PLC isoform, and its sensitivity to PLA2 was negligible due to steric hindrance at the sn-2 position. In contrast, the C_(12)-spacered PyroC_(12)-PtdEtn-BHQ demonstrated a remarkable selectivity for PLA2 and the best relative PLA2/PLC sensitivity, significantly outperforming previously known probes'. These results open an avenue for future in vivo experiments and for new probes to detect PL activity.